The overall goal of the previous cycle of this BRP project has been to improve the spatial resolution of functional Magnetic Resonance Imaging (fMRI) and optical technology in order to bridge the gap between macroscopic fMRI and the underlying microscopic electrical activity of single neurons and dilation and constriction of single blood vessels. To achieve this goal, we have developed and experimentally validated an integrated suite of technologies providing improved sensitivity to the observable physiological and biophysical parameters, with high spatial and temporal resolution, to measure direct and indirect consequences of brain neuronal activity at multiple scales. We successfully applied the new tools to study the relationship between imaging signals and the underlying neuronal activity in healthy brain and under clinical conditions such as cortical spreading depression and stroke. Of particular note, data acquired during the previous funding cycle, along with data from others, increasingly suggest that differential vascular control originates from activation of distinct neuronal sub-populations through release of specific vasoactive agents. Thus, the main goal of this renewal is to combine the BRP technological developments with recent revolutionary methods in genetic labeling and remote control of neuronal activity allowing targeted activation of identified neuronal cells and cellular populations to identify macro- and microscopic hemodynamic signatures of activation in populations of neurons with known phenotype and neurotransmitter content. Specifically, we will directly control firing patterns in labeled single cells and cellular populations, while measuring the resultant vascular, metabolic and neuronal response in normal and genetically-modified rats and mice across spatial scales. The proposed experiments will provide important insights into pathways of neurovascular communication and will identify vasoactive messengers, release of which drives stimulus- evoked hemodynamic signals. While targeted activation of identified neuronal phenotypes was not available when our BRP was originally funded, today we are in a unique position to combine these novel genetic tools with a suite of imaging technologies that have been developed during the previous funding cycle. The experimental measurements will be integrated within a comprehensive modeling framework that relates physiological parameters and imaging observables at the microscopic and macroscopic scales. This combines 1) bottom-up models that enable prediction of macroscopic imaging signals from microscopic measures of vessel diameters, flow, and the intravascular and extravascular oxygenation state; and 2) top-down models that enable estimation of physiological variables of interest (primarily cerebral blood flow and O2 metabolism) from non-invasive Blood Oxygenation Level Dependent (BOLD) and Arterial spin Labeling (ASL) fMRI experiments. This work will greatly extend the utility of fMRI as a quantitative probe of physiology for both basic and clinical basic neuroscience applications.